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L-homoserine tolerant escherichia coli and application thereof

A technology of Escherichia coli and homoserine, applied in the fields of application, bacteria, carbon-carbon lyase, etc., can solve the problems of insufficient yield, low conversion rate, unfavorable low-cost production, etc.

Pending Publication Date: 2022-01-21
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing reports have shortcomings such as low conversion rate, poor homoserine tolerance, insufficient yield, and unfavorable large-scale and low-cost production.

Method used

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  • L-homoserine tolerant escherichia coli and application thereof
  • L-homoserine tolerant escherichia coli and application thereof
  • L-homoserine tolerant escherichia coli and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1, ARTP mutagenesis improves the tolerance of Escherichia coli to L-homoserine

[0079] 1. ARTP mutagenesis

[0080] L-homoserine is toxic to cell growth. When carrying out inorganic salt fermentation, high concentration of L-homoserine will lead to the inability to further increase the biomass, thereby limiting the yield of the product. Through the method of ARTP mutagenesis, the tolerance of Escherichia coli BW25113 to high concentration of L-homoserine was improved.

[0081] Spread the BW25113 bacterial solution cultured for 12 hours evenly on a sterile glass slide. When using ARTP to irradiate, the irradiation time is 30s, 60s, 90s, 120s, 150s respectively. After the irradiation, put the slide glass into 1 mL of sterile saline, shake and wash it out, take 100 microliters of liquid to dilute and spread on different petri dishes. After culturing at 37°C for 3 days, a single clone was picked and streaked on a Petri dish containing 40g / L L-homoserine. After...

Embodiment 2

[0085] Embodiment 2, the construction of chassis bacterial strain HS03

[0086] Using Crispr / cas9 technology to knock out, replace or insert genes on the chromosome of HS02 CGMCC No.21837, the construction of the chassis strain HS03 mainly includes: knocking out the threonine metabolism pathway homoserine kinase thrB, knocking out the methionine metabolism bypass homoserine amber Acyltransferase metA, knockout of lysine metabolic bypass diaminoheptanoate decarboxylase lysA, knockout of DNA-binding transcriptional repressor lacI, knockout of glyoxylate pathway inhibitory protein iclR, overexpression of phosphoenolpyruvate carboxylation Enzyme ppc, knockout of soluble pyridine nucleotidyl transferase sthA, overexpression of pyridine nucleotidyl transferase pntAB, as follows:

[0087] The pCas and pTargetF plasmids in the following examples are described in the following reference "Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System; Applied Environmental ...

Embodiment 3

[0131] Example 3, Construction of L-homoserine strain from oxaloacetate to aspartic acid pathway

[0132] 1. Deletion of pyruvate kinase IpykF on the basis of HS03 strain (GeneBANK: QKN73339.1, submission date 20200710)

[0133] The pCas plasmid (kanamycin resistance) was introduced into HS03. Because the pCas plasmid is a temperature-sensitive plasmid, cultured at 30°C for 15 hours, and the positive clone HS03 / pCas was screened.

[0134] Using pTargetF as a template and pTargetF-pykF-F / R as primers, PCR amplification was carried out to obtain a 2118bp plasmid containing pykF gene recognition N20, which was named pTargetF-pykF after sequencing and verification. The plasmid contained the coding of pykF gene gRNA Gene, the nucleotide sequence of N20 is CGAAGCCTCTGACGGCATCA.

[0135] Using BW25113 genomic DNA as a template, pykF-up-F / R and pykF-down-F / R primers were amplified to obtain a 500bp upstream homology arm of the pykF site and a 500bp downstream homology arm of the pykF...

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Abstract

The invention discloses L-homoserine tolerant escherichia coli and an application thereof. The L-homoserine tolerant escherichia coli provided by the invention is escherichia coli HS02 CGMCC No. 21837. The invention also provides a recombinant bacterium which is obtained by modifying two metabolic pathways in the chassis host bacterium and matching the metabolic flux of the two metabolic pathways according to a ratio of 1: 1. The two metabolic pathways are as follows: aspartic acid is synthesized from oxaloacetic acid and then L-homoserine is obtained; the aspartic acid is synthesized from fumaric acid, and then L-homoserine is obtained; the chassis host bacteria are obtained by reducing the activities of lacI, metA, lysA, thrB, stA and iclR or the expression quantity of encoding genes of the lacI, metA, lysA, thrB, stA and iclR and improving the activities of ppc and pntAB or the expression quantity of the encoding genes of the ppc and pntAB in starting bacteria with L-homoserine tolerance and an aspartic acid-threonine pathway. According to the L-homoserine high-level fermentation engineering strain obtained by the invention, the fermentation intensity of the L-homoserine can be up to 2.10 g / L / h.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an L-homoserine-tolerant Escherichia coli and its application. Background technique [0002] L-homoserine is a non-proteinogenic amino acid and is a precursor for the synthesis of important L-aspartate-derived essential amino acids such as L-methionine, L-threonine and L-isoleucine. L-homoserine has shown great potential application prospects in food, medicine, cosmetics, agriculture and animal feed industries. Furthermore, L-homoserine is a promising platform compound that can be used to produce products such as O-acetyl-homoserine, o-succinyl-L-homoserine, 1,3-propanediol, isobutanol, and γ-butyrolactone and other fine chemicals. Therefore, it has aroused great interest and the market demand is constantly increasing. At present, there are three main production methods of L-homoserine: chemical method, enzyme catalysis and microbial fermentation. Microbial fermentation has attra...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/21C12N15/70C12N15/31C12N15/54C12N15/60C12P13/06C12R1/19
CPCC12N9/1029C12N9/88C12N9/1205C12N9/0036C12N9/1096C12N9/0016C07K14/245C12N15/70C12N15/52C12P13/06C12Y203/01046C12Y401/0102C12Y207/01039C12Y401/01031C12Y106/01002C12Y106/01C12Y403/01001C12Y206/01021C12Y104/01002Y02A50/30
Inventor 牟庆璇毛贤军陶勇于波
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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